Laryngeal verrucous carcinoma: A clinicopathologic study and detection of human papillomavirus using polymerase chain reaction

Laryngeal verrucous carcinoma (LVC) is a rare, well-differentiated variant of squamous carcinoma with a low malignant potential. Human papillomavirus (HPV)–16 DNA has been identified in a small number of LVC and an etiologic relationship has been suggested. A correlative clinical and molecular pathological study was performed in order to determine the prevalence and typing of HPV DNA in LVC. Possible associations between patient and tumor subsets, and the presence of HPV DNA were also investigated. Formalin-fixed, paraffin-embedded tissue samples from 29 patients with LVC were examined by polymerase chain reaction (PCR) using DNA primers specific for HPV types 6b/11, 16, and 18. Overall, HPVDNA was detected in 13 (45%) of the cases. Of these, HPV-16 DNA, HPV-18 DNA, and both HPV-16 DNA and HPV-18 DNA were detected in 4 (14% overall; 31% of positive cases), 4, and 5 (17% overall; 38% of positive cases), respectively. HPV-6b/11 DNA was not detected in any LVCs. In 16 cases, no HPV DNA was detected. There was a trend toward HPV DNA detection in higher stage tumors. HPV DNA detection was unrelated to patient age, tumor site, or radiotherapeutic responsiveness. The detection of HPV DNA in 45% of LVCs suggests an association between the presence of HPV-16 DNA and HPV-18 DNA, and some LVCs.     

General primer-mediated polymerase chain reaction for simultaneous detection and typing of human papillomavirus DNA in laryngeal squamous cell carcinomas

The aim of this study was to investigate the prevalence of different human papillomavirus (HPV) types in laryngeal squamous cell carcinomas using general primer-mediated polymerase chain reaction (PCR). Tumour sections from 42 patients with laryngeal carcinomas were investigated. For HPV DNA amplification, consensus primers were used which were directed to the LI coding region of the HPV genome. Analysis of the PCR products was done using 2% agarose gel electrophoresis followed by restriction enzyme analysis to identify different HPV types. Amplification of the human TGF-β DNA was successfully performed in 36/42 (85.7%) of samples confirming the presence of sufficient DNA for viral amplification. HPV DNA was detected in 8/36 (22.2%) of the tumours examined (three HPV-6, two HPV-16, one HPV-11, two unknown HPV types). HPV DNA was not detected in any of the non-neoplastic laryngeal mucosa which was used as control (n = 15). Fifty per cent of women had HPV-positive tumours compared with 8% of men (x²= 5.8, P<0.05). Our data indicate that while the overall prevalence of HPV in laryngeal carcinomas is fairly high (22.2%), the frequency of high-risk types (HPV-16 & HPV-18) is low (5.5%). HPV probably acts as a promoter in the multistep process of carcinogenesis in squamous mucosal cells of the larynx.     

Molecular detection and typing of human papillomavirus in laryngeal carcinoma specimens

Conclusions. Human papillomavirus (HPV) DNA was detected in 32% of laryngeal carcinoma biopsy samples studied. The genotypes identified were high-risk types, the most frequent being HPV 16. Viral DNA was integrated into the host genome (genotype HPV 16), providing supporting evidence for a role of HPV in the carcinogenic pathway of laryngeal squamous cell carcinoma. Objective. HPV has been detected in laryngeal lesions, both benign and neoplastic, with a variable frequency (8–60%). These viral agents have been proposed as an adjuvant or cofactor in head and neck carcinogenesis because of their oncogenic properties. The aims of this study were to identify HPV in laryngeal carcinoma samples and to describe the physical state of the viral genome, i.e. its integration to the host DNA. Material and methods. Formalin-fixed, paraffin wax-embedded tumor samples from patients with newly diagnosed laryngeal carcinomas were collected. The HPV genome was identified using polymerase chain reaction (PCR) with primers complementary to the conserved region L1 (MY09-11). Genotyping was accomplished by restriction fragment length polymorphism. Samples positive for HPV 16 were assayed by PCR with primers complementary to region E2, interrupted during viral genome integration. Results. Ten of the 31 samples (32%) were positive for HPV DNA and all of the samples were positive for human β-globin. The genotypes identified were HPV 16 (n=3), HPV 58 (n=2) and HPV 39, 45, 51, 59, 66 and 69 (n=1 for each). The three samples positive for HPV 16 had lost region E2, meaning that the viral DNA had been integrated into the host genome.     

Molecular detection and typing of human papillomavirus in laryngeal carcinoma specimens

CONCLUSIONS: Human papillomavirus (HPV) DNA was detected in 32% of laryngeal carcinoma biopsy samples studied. The genotypes identified were high-risk types, the most frequent being HPV 16. Viral DNA was integrated into the host genome (genotype HPV 16), providing supporting evidence for a role of HPV in the carcinogenic pathway of laryngeal squamous cell carcinoma. OBJECTIVE: HPV has been detected in laryngeal lesions, both benign and neoplastic, with a variable frequency (8-60%). These viral agents have been proposed as an adjuvant or cofactor in head and neck carcinogenesis because of their oncogenic properties. The aims of this study were to identify HPV in laryngeal carcinoma samples and to describe the physical state of the viral genome, i.e. its integration to the host DNA. MATERIAL AND METHODS: Formalin-fixed, paraffin wax-embedded tumor samples from patients with newly diagnosed laryngeal carcinomas were collected. The HPV genome was identified using polymerase chain reaction (PCR) with primers complementary to the conserved region L1 (MY09-11). Genotyping was accomplished by restriction fragment length polymorphism. Samples positive for HPV 16 were assayed by PCR with primers complementary to region E2, interrupted during viral genome integration. RESULTS: Ten of the 31 samples (32%) were positive for HPV DNA and all of the samples were positive for human beta-globin. The genotypes identified were HPV 16 (n=3), HPV 58 (n=2) and HPV 39, 45, 51, 59, 66 and 69 (n=1 for each). The three samples positive for HPV 16 had lost region E2, meaning that the viral DNA had been integrated into the host genome.     

The quantitative analysis of the human papillomavirus DNA load in submandibular gland lesions with droplet digital polymerase chain reaction

Background: The relationship between infection with human papillomavirus (HPV) and tumorigenesis of salivary gland remains controversial.

Objectives: This study explored the relationship between HPV and salivary gland lesions as well as that of the HPV infection status and p16^INK4A immunoreactivity. The HPV DNA loads were also quantitatively evaluated.

Materials and Methods: Tissue samples from 31 submandibular gland lesions were evaluated. p16^INK4A immunohistochemical (IHC) staining, nested polymerase chain reaction (PCR), DNA sequencing, and droplet digital PCR (ddPCR) were performed.

Results: Non-neoplastic lesion, benign tumors, and malignant tumors were noted in 9, 16, 6 cases, respectively. p16^INK4A immunoreactivity was higher in malignant tumors than in benign tumors (50.0% vs. 6.3%). Single PCR with MY09/11 found that all samples were negative. Nevertheless, nested PCR revealed a high HPV-DNA positivity rate of 96.8%. No relationship between the HPV status and p16^INK4A immunoreactivity was shown. HPV-18 was the only subtype identified in this study. ddPCR showed significantly lower HPV-18 DNA loads in submandibular gland lesions than in oropharyngeal cancers.

Conclusions: HPV-DNA positivity and p16^INK4A-immunoreactivity were not correlated in submandibular gland lesions. The loads of HPV DNA detected in this study were small. HPV positivity therefore may not be associated with tumorigenesis of the submandibular gland.     

Detection of human papillomavirus DNA in hypopharyngeal carcinoma]

The detection of human papillomavirus type-16 and -6b was performed in 28 cases of hypopharyngeal squamous cell carcinomas by polymerase chain reaction. Human papillomavirus (HPV) DNAs extracted from paraffin-embedded metastatic lymph node tissue were used for polymerase chain reaction with amplification of the E1 region of HPV genotype 6b and the E6 region of HPV genotype 16. HPV type 6b sequences were detected in three cases; positive amplification of type 16 sequences was also seen in three cases. These findings suggest that HPV infection might be closely associated with the development of some hypopharyngeal carcinomas as it is with laryngeal and uterine cervical carcinogenesis.     

Short-fragment PCR assay for highly sensitive broad-spectrum detection of human papillomaviruses in laryngeal squamous cell carcinoma and normal mucosa: clinico-pathological evaluation

The aim of the study was to determine the prevalence and genotypes of HPV infection in laryngeal cancer specimens, normal mucosa obtained from the surgical margin and laryngeal nodules using a novel high sensitive and specific SPF₁₀ HPV DNA test, PCR/DEIA method and INNO-LiPA genotyping assay. The correlation between HPV presence and clinico-pathological features was analyzed. Tissue samples were collected from 93 primary laryngeal squamous cell carcinoma (LSCC), 49 specimens of normal mucosa and from 22 specimens of laryngeal nodules serving as control group. HPV DNA was amplified by the short PCR fragment (SPF₁₀) primer set using HPV DNA enzyme immunoassay (DNA/DEIA) method and INNO-LiPA HPV genotyping assay. Human papillomavirus was detected in 33 (35.5%) of the 93 samples from LSCC, in 4 (8.2%) of 49 samples of the normal mucosa and it was not detected in any of the sample from the control group. Twenty-eight of 33 (81.8%) were positive for HPV-16, 6 of 33 (18.2%) were positive for HPV-18 and 5 of 33 (15.1%) were positive for HPV-33. Multiple infection was found in 5 of 33 (15.1%); 3 samples were positive for HPV-16 and HPV-33, 2 samples for HPV-16 and HPV-18. There was a statistically significant correlation between the presence of HPV in LSCC tumors and in control group samples and between the presence of HPV in the tumors and normal mucosa from the free surgical margin. The presence of HPV infection in 35.5% of the cases suggests a possible role in the etiology of laryngeal cancer and supports the role of high-risk types of HPV (16, 18 and 33) in LSCC. HPV infection is not likely to influence survival rates as an independent prognostic factor in patients with laryngeal cancer.     

Detection of human papillomavirus type 16 and 18 in laryngeal cancer using PCR methods

Human papillomavirus (HPV) is a small DNA virus. HPV is can be divided into two groups: mucosal and cutaneous. HPV have various oncogenic potential. The correlation between presence of high oncogenic type of HPV and carcinogenesis was confirmed in human anogenital tract and cervical carcinoma. The latest studies suggest HPV infection involvement in benign and malignant lesions of aerodigestive tract. The aim of our study was to determine the presence of the HPV genes E6/E7. The specimens were taken after total laryngectomy from 20 patients with squamous cell carcinoma. The fresh tissue specimens were frozen at -70 degrees C until DNA extraction and HPV detection. The presence of HPV DNA was assessed by polymerase chain reaction using specific primers for HPV 16 and 18 E6/E7. There were 7 (35%) HPV positive specimens in our group. 5 (25%) specimens were HPV 16 positive, 4 (20%) specimens were HPV-18 positive. In two cases HPV 16 and HPV 18 was present. The results suggest that high oncogenic types of HPV may play a role in pathogenesis of laryngeal carcinoma.     

Human papillomavirus types 11 and 16 detected in nasopharyngeal carcinomas by the polymerase chain reaction

Most nasopharyngeal carcinomas (NPCs) are of the nonkeratinizing or undifferentiated types, which are consistently associated with Epstein-Barr virus (EBV). The smaller group of highly differentiated, keratinizing NPCs seems to be only infrequently associated with EBV. In order to examine whether these rare tumors were related to another oncogenic virus, the authors used the polymerase chain reaction to examine paraffin-embedded sections of 15 keratinizing NPCs for human papillomavirus (HPV) types 6, 11, 16, and 18 genomic sequences. HPV DNA was found in 4 tumors (1 HPV-11-positive, and 3 HPV-16–positive tumors). None of 23 undifferentiated or nonkeratinizing NPCs harbored HPV DNA. The putatively oncogenic HPV type 16 may thus be involved in the carcinogenesis of some EBV-negative keratinizing squamous cell nasopharyngeal carcinomas.     

Prevalence and clinical features of human papillomavirus in head and neck squamous cell carcinoma in Okinawa,southern Japan

Previous studies from Okinawa, a subtropical island in southern Japan, demonstrated a higher prevalence of human papillomavirus (HPV) in oral carcinoma and a higher incidence of oral and pharyngeal carcinoma than those for mainland Japan. The present study aims to investigate epidemiologic and clinical features of HPV in head and neck squamous cell carcinoma (HNSCC) in Okinawa. A total of 150 DNA samples from 150 Okinawan patients with head and neck squamous cell carcinoma (HNSCC) were screened for HPV sequences by PCR using three consensus primer sets, and HPV types were determined by direct sequencing. The samples were consisted of 46 cases from the hypopharynx, 44 from the oropharynx, 16 from the larynx, 25 from the oral cavity, 10 from the maxillary sinus, and 9 from the nasopharynx. HPV DNA was detected in 45 (30.0%) HNSCCs, and HPV-16 was identified in 86.7% of positive specimens. The highest prevalence of the HPV sequence was found in oropharyngeal carcinomas (50.0%), especially in tonsillar cancer (63.6%). Multivariate analysis showed that oropharyngeal carcinoma (P = 0.002; OR = 5.34; 95% CI = 1.83–15.58), oral cavity carcinoma (P = 0.012; OR = 4.94; 95% CI = 1.43–17.10), and histological poor differentiation (P = 0.011; OR = 4.25; 95% CI = 1.39–13.04) each independently increased the prevalence of HPV infection. The present study reveals that patients with HNSCC, e.g., oropharyngeal and oral cavity carcinomas, in Okinawa have relatively high HPV-16 positive rates and low HPV-18 positive rates comparing with mainland Japan.     

Detection and typing of human papillomavirus in verrucous carcinoma of the oral cavity using the polymerase chain reaction

OBJECTIVE: This study examined the prevalence and types of human papillomavirus (HPV) DNA in oral cavity verrucous carcinoma. DESIGN: This was of a retrospective screening study. Formalin-fixed, paraffin-embedded tissue samples were examined by the polymerase chain reaction using DNA primers specific for HPV types 6b/11, 16, and 18. SETTING: The majority of patients were seen at referral centers in Ontario, Canada. PATIENTS: This study examined 29 oral cavity verrucous carcinomas occurring in a sample of 25 patients from four institutions between 1966 and 1992. All tumors met standardized histologic diagnostic criteria of verrucous carcinoma. MAIN OUTCOME MEASURE: The prevalence of HPV 6b/11, 16, and 18 DNA was determined by the PCR technique. RESULTS: The HPV DNA was detected in 12 (48%) of 25 patients. The HPV 6b/11 DNA, HPV 16 DNA, HPV 18 DNA, and HPV 16 DNA plus HPV 18 DNA, were detected in one (4%), one (4%), nine (36%), and one (4%) cases, respectively. CONCLUSIONS: The detection of HPV 18 DNA in 40% of oral cavity verrucous carcinomas suggests an association between the presence of HPV 18 DNA and some oral cavity verrucous carcinomas. The etiologic and prognostic significance of HPV 18 for oral cavity verrucous carcinoma remains unanswered and will require further study.     

Detection and Typing of Human Papillomavirus DNA in Benign and Malignant Tumours of Laryngeal Epithelium

The role of human papillomaviruses (HPV) in laryngeal squamous cell carcinoma has not yet been established. Thirty-three cases of laryngeal squamous cell carcinoma were analysed for the presence of HPV DNA and compared with 25 cases of normal larynx and 29 cases of laryngeal squamous papilloma in their positivity index. The presence of HPV DNA was analysed by using L1 consensus primers and also by primers specific for the E7 gene of HPV types 16 and 18. Four normal laryngeal samples (16%) were positive for HPV DNA against the 24 samples (82%) (p&lt;0.001) found for laryngeal papilloma and 16 (48.5%) (p&lt;0.05) found for laryngeal squamous cell carcinoma. HPV 16 was the type most frequently found in laryngeal carcinoma samples. Our results support an etiologic role for this type of HPV in the pathogenesis of laryngeal carcinoma.     

Detection of HPV-16-related DNA sequence in laryngeal squamous cell carcinoma]

The gene libraries of laryngeal squamous cell carcinoma (LSCC) and laryngeal papilloma (LP) were screened by hybridization analysis with alpha-32P-labelled HPV-16 DNA under low stringency (Tm = 40 degrees C). It showed that the relative sequences were detected in LSCCs (11/16, 68.8%), but not in LP. The HPV-16-related DNA harbored in negative hybridization lesions were further analysed by polymerase chain reaction to amplify E6/E7 gene of HPV-16-related DNA. The results showed that 1/5 LSCCs and 2/2 LPs, were positive. The data suggest that development of LSCC should be related to the HPV infection, and HPV may be one of the inducers of LSCC.     

Relationship of Human Papillomavirus to Schneiderian Papillomas

Objectives/Hypothesis: To classify a large group of Schneiderian papillomas (SPs) into their histologic subtypes and to determine the incidence of human papillomavirus (HPV) in each subtype. Study Design: Pathologic review and polymerase chain reaction–based (PCR-based) examination of archived tissue. Methods: Slides of 114 tumors diagnosed as Schneiderian, inverting, fungiform, or cylindric cell papillomas, or any associated carcinomas, were examined by a head and neck pathologist. Using PCR, consensus primers for the L1 region of HPV were used to determine the presence of HPV in the tumors. This was also performed on normal turbinate control specimens. Results: Eighty-two (78%) were the inverting subtype, 21 (20%) the fungiform subtype, and 2 (2%) the cylindric cell type. Nine tumors were diagnosed as either verrucous or squamous cell carcinoma. Eighty-eight percent of the tumors had DNA of sufficient quality to be amplified using PCR. Of these, 5 of 69 (6.8%) inverting, 17 of 17 (100%) fungiform, and 0 of 2 cylindric cell papillomas were positive for HPV. One of nine (11.1%) cancers was positive for HPV. No normal turbinate tissue contained HPV. HPV types 6b and 11 accounted for all cases of fungiform papillomas. Of the five HPV-positive inverting papillomas, three had HPV type 11 and two had HPV type 16. The single carcinoma containing HPV contained HPV type 18. Conclusions: The histologic subtype of SPs is important, as their etiologies appear to be different. HPV 6b and 11 appear to be involved in all cases of fungiform papillomas but are only rarely involved in cases of inverting or cylindric cell papillomas. HPV 16 may rarely play a role in cases of inverting papillomas, and HPV 16 and 18 may be involved in a subset of cases of carcinomas originating in an inverting papilloma.     

Identification of HPV DNA in patients with juvenile-onset recurrent respiratory papillomatosis using SYBR Green real-time PCR

OBJECTIVE: Recurrent respiratory papillomatosis (RRP) is the most common benign neoplasm affecting the larynx and upper respiratory tract in children. Human papillomavirus (HPV) has been implicated as the cause of RRP, most commonly types 6 and 11. The present study was undertaken to evaluate the occurrence of HPV types in a group of patients with juvenile-onset RRP (JORRP). METHODS: The study group consists of 23 patients with JORRP. The clinical records of the patients were reviewed, and JORRP was classified as non-aggressive or aggressive. The laryngeal biopsies were taken and investigated for HPV DNA presence using real-time polymerase chain reaction (PCR) with a set of consensus primers (MY09/11). Viral typing was subsequently performed by real-time PCR with type-specific primers for HPV types 6, 11, 16, 18, 31, and 33. RESULTS: HPV presence was detected in all samples with amplifiable DNA. HPV-11 was revealed in 61.9% of the patients and HPV-6 in 23.8%. Double positivity for HPV types 6 and 11 was identified in 14.3%. Our findings suggest that RRP runs a more aggressive clinical course when HPV-11 infection is present (p=0.0265). CONCLUSIONS: Our results suggest a high frequency of HPV infection in the upper respiratory tract of the studied patients. We believe that the routine application of molecular techniques such as PCR for detection and analysis of HPVs in patients with RRP has diagnostic and prognostic significance.     

Human papillomavirus and infiltration of CD8- and Foxp3-positive immune cells in sinonasal inverted papillomas

Abstract

Background: Sinonasal inverted papilloma (IP) is a benign tumor with a high risk of local recurrence and a potential to malignify and Human papillomavirus (HPV) has been suggested an etiological factor. p16INK4a (p16) overexpression is considered a surrogate marker for HPV, but whether p16 and HPV correlate to IP is uncertain. Besides, a prognostic role of tumor infiltrating lymphocytes (TILs) are observed in many tumors, however their role in IP is sparsely studied.

Aims/objectives: We hence analyzed IPs for the presence and the prognostic role of HPV and p16 overexpression together with CD8+ and FoxP3+ TILs in a population-based study.

Material and methods: 98 IP patients diagnosed 2001–2010 were identified from the Swedish Cancer Registry and analyzed for HPV by PCR and p16, CD8 and FoxP3 was by immunohistochemistry.

Results: In total, 12.2% of the IPs were HPV-positive (nine HPV-11, two HPV-6 and one HPV-45). Patients with HPV-positive lesions were younger (p?=?.003) and tended to present with more dysplasia. No correlation was observed between TILs and prognosis.

Conclusions and significance: Our data suggests that patients with HPV-positive IPs present with different clinical characteristics, suggesting possibly different disease entities. Moreover, recurrences may occur >5?years, which should be considered in the follow-up.     

Coinfection of HPV-11 and HPV-16 in a Case of Laryngeal Squamous Papillomas With Severe Dysplasia

Human papillomavirus (HPV) types 6 and 11 have been associated with benign laryngeal papilloma, while HPV-16 is occasionally associated with laryngeal carcinoma. In this study, a case of laryngeal squamous papillomas with severe dysplasia was evaluated for the presence of HPV infection. The biopsy specimens were taken from a 58-year-old female patient at two different time points 3 months apart. Architecturally, the tumor showed papillary configuration reminiscent of squamous papilloma. Cytologically, the lesion showed morphologic features characteristic of severe squamous epithelial dysplasia. HPV infection was determined by DNA in situ hybridization using type-specific HPV-DNA probes. HPV-11 probes demonstrated homogeneous nuclear staining, suggesting productive viral replication. In contrast, HPV-16 probe produced a speckled pattern, suggesting HPV-16 DNA integration. Normal laryngeal epithelium did not yield specific hybridization. The presence of HPV-11 and HPV-16 was confirmed by PCR using HPV type-specific primers. Immunocytochemical staining was performed to detect Ki-67, a proliferation marker, and p53. Ki-67 expression was demonstrated throughout the whole thickness of epithelium. Staining for p53 was negative. This study suggests that multiple HPV infections can occur in the same lesion and that HPV-16 infection and its DNA integration may contribute to the occurrence of severe dysplasia in the lesion described.     

Verrucous carcinoma of the larynx. Possible human papillomavirus etiology

Verrucous carcinoma of the larynx is a distinct and uncommon variant of well-differentiated squamous cell carcinoma. By DNA hybridization techniques, we clearly demonstrated human papillomavirus (HPV-16-related) sequences in five patients with this neoplasm. In addition, HPV-16-related sequences were found in adjacent normal tissues. The DNAs from squamous cell carcinomas of the larynx were negative when hybridized to HPV-6, -11, or -16. Postirradiation anaplastic transformation of verrucous carcinoma has been described. We believe that radiotherapy should not be given unless the potential consequences are fully explained because of its potential to activate or alter HPV-16-related sequences.     

Human papillomavirus in sinonasal papillomas and squamous cell carcinoma.

The diagnostic and prognostic relevance of human papillomavirus (HPV) types 6, 11, 16, and 18 in squamous papilloma, inverted papilloma, and squamous carcinoma of the sinonasal epithelium was examined using the polymerase chain reaction (PCR) technique. Four (15%) of 26 squamous papillomas, 7 (24%) of 29 inverted papillomas, and 1 (4%) of 24 squamous carcinomas were positive for HPV when examined using the PCR amplification technique. Human papillomavirus 6 was present in 5 specimens (3 squamous and 2 inverted papillomas); HPV-11 was present in 6 specimens (1 squamous and 5 inverted papillomas); and HPV-18 was present in 1 of 24 squamous carcinomas. HPV-16 was not identified in any specimen. The proportion of tissue samples showing HPV presence, and the association of HPV types 6 and 11 with benign lesions and HPV-18 with malignant lesions, are both in accord with findings from prior investigations. Two major questions regarding nasal papilloma are the probability for lesion recurrence after surgical excision and the risk for malignant transformation. There is no unanimity of opinion regarding the prognostic value of histopathologic dysplasia to forecast these outcomes. HPV is etiologically related to a subset of sinonasal papillomas and squamous carcinoma, and those with benign and malignant clinical course are separable on basis of HPV type. Because of the paucity of these nasal lesions, a multi-institutional prospective collaborative study is the ideal way to address these questions.